The exposure of phosphatidylserine (PS) and other aminophospholipids (aminoPL) on the surface of activated or injured blood cells and endothelium is thought to play a key role in the initiation and regulation of blood coagulation. De novo surface exposure of aminophospholipids has also been implicated in the activation of both complement and coagulation systems after tissue injury, and in removal of injured or apoptotic cells by the reticuloendothelial system. Although migration of these phospholipids (PL)from inner-to-outer plasma membrane leaflets is known to be triggered by elevated intracellular [Ca.sup.2+ ] ([Ca.sup.2+ ].sub.i) and to be associated with vesicular blebbing of the cell surface, little is known about the cellular constituents that participate in this process.
Role of Cell Surface PS in Coagulation
Several enzyme complexes of the coagulation cascade require assembly on a receptive membrane surface for full expression of catalytic activity (K. G. Mann, et al., Annu. Rev. Biochem. 57:915-956, 1988; S. Krishnaswamy, et al., J. Biol. Chem. 267:26110-26120, 1992; P. B. Tracy, Semin. Thromb. Hemost. 14:227-233, 1988). In the case of the tenase (FVIIIaFIXa) and prothrombinase (FVaFXa) complexes, this surface catalytic function of the plasma membrane is not normally expressed by quiescent cells, but is rapidly induced upon cell activation (in platelets) or upon cell injury (in platelets, endothelium and other cells) (E. M. Bevers, et al., Blood Rev. 5:146-154, 1991; J. Rosing, et al., Blood 65:319-332, 1985; E. M. Bevers, et al., Eur. J. Biochem. 122:429-436, 1982; E. M. Bevers, et al., Biochim. Biophys. Acta 736:57-66, 1983; T. Wiedmer, et al., Blood 68:875-880, 1986). Although specific cell surface protein receptors for FVa and FVIIIa have been postulated, these factors show specific avidity for PS-containing liposomes, and in cell-free systems, this lipid alone can support the catalytic function of the prothrombinase and tenase enzymes (J. Rosing, et al., supra, 1985; M. E. Jones, et al., Thromb. Res. 39:711-724, 1985; G. E. Gilbert, et al., Biochemistry 3 2:9577-9585, 1993; G. E. Gilbert, et al., J. Biol. Chem. 265:815-822, 1990; G. E. Gilbert, et al., J. Biol. Chem. 267:15861-15868, 1992). We and others have shown that PS rapidly moves to the surface of plasma membrane upon platelet stimulation, and that this exposure of PS correlates with expression of the platelet's FVa & FVIIIa binding sites and expression of surface catalytic function for tenase and prothrombinase (P. Williamson, et al., Biochemistry 31:6355-6360, 1992; F. Basse, et al., Biochemistry 32:2337-2344, 1993; C.-P. Chang, et al., J. Biol. Chem. 268:7171-7178, 1993; J. Connor, et al., Biochim. Biophys. Acta 1025:82-86, 1990; P. Comfurius, et al., Biochim. Biophys. Acta. 1026:153-160, 1990). Smeets, et al., Biochem. Biophys. Acta Biomembr. 1195:281-286, 1994, Williamson, et al., Biochem. 34:10448-10455, 1995; Bratton D. L., J. Biol. Chem. 269:22517-22523, 1994). Additional evidence that surface-exposed PS provides the physiological receptor site for these enzyme complexes is provided by (1) the capacity of PS-containing liposomes or phosphoserine to compete binding of FVIIIa to activated platelets (G. E. Gilbert, et al., J. Biol. Chem. 266:17261-17268, 1991), (2) the capacity of annexin V and other proteins with affinity for membrane PS to mask the FVa and FVIIIa binding sites expressed by activated platelets (P. Thiagarajan, et al., J. Biol. Chem. 265:17420-17423, 1990; P. Thiagarajan, et al., J. Biol. Chem. 266:24302-24307, 1991; J. Dachary-Prigent, et al., Blood 81:2554-2565, 1993; J. Sun, et al., Thromb. Res. 69:289-296, 1993); (3) evidence that platelets congenitally deficient in inducible FVa and FVIIIa receptors are also defective in stimulated exposure of membrane PS ("Scott syndrome"; see below) (J. P. Miletich, et al., Blood 54:1015-1022, 1979; J. Rosing, et al., Blood 65:1557-1561, 1985; P. J. Sims, et al., J. Biol. Chem. 264:17049-17057, 1989; S. S. Ahmad, et al., J. Clin. Invest. 84:824-828, 1989; F. Toti, et al., Blood 87:1409-1415, 1996). In addition to the catalytic function PS provides to the prothrombinase and tenase complexes, surface exposed aminophospholipids have been shown to promote the activities of the tissue factor-FVIIa and protein S-activated protein C enzyme complexes of the coagulation system, as well as the activity of the alternative pathway C3-convertase (C3bBb enzyme complex) of the complement system (W. Ruf, et al., J. Cell. Biol. 266:2158-2166, 1991; F. J. Walker, J. Biol. Chem. 256:11128-11131, 1981; R. H. Wang, et al., J. Clin. Invest. 92:1326-1335, 1993; P. F. Neuenschwander, et al., Biochemistry 34:13988-13993, 1995).
In addition to the central role that inducible expression of plasma membrane PS is thought to play in the platelet hemostatic response, the surface exposure of PS and phosphatidylethanolamine (PE) in response to membrane injury has been implicated in a variety of thrombotic and inflammatory disorders. For example, repeatedly sickled SS hemoglobin erythrocytes exhibit increased surface exposure of PS, which promotes prothrombinase assembly and accelerates plasma clotting in vitro, and may contribute to thrombotic complications that can arise in sickle cell disease (P. F. Franck, et al., J. Clin. Invest. 75:183-190, 1985; N. Blumenfeld, et al., Blood 77:849-854, 1991). Increased PE exposure on sickled RBCs (and other cells) has also been shown to promote complement activation with resulting accumulation of C3b/C3d and C5b-9 on the cell surface, potential factors contributing to the accelerated clearance and increased fragility of these cells (R. H. Wang, et al., supra, 1993). PS exposure secondary to immune injury to the endothelium has also been implicated in the thrombo-embolic complications of hyperacute graft rejection, and PS exposure secondary to C5b-9 accumulation on platelets and red cells has been suggested to contribute to the high risk of venous thrombosis in Paroxysmal Nocturnal Hemoglobinuria (J. L. Platt, et al., Immunol. Today 11:450-6; discuss, 1990; A. P. Dalmasso, Immunopharmacology 24:149-160, 1992; A. P. Dalmasso, et al., Am. J. Pathol. 140:1157-1166, 1992; T. Wiedmer, et al., Blood 82:1192-1196, 1993, K. K. Hamilton, et al., J. Biol. Chem. 265:3803-3814, 1990; S. P. Kennedy, et al., Transplantation 57:1494-1501, 1994)). In the "antiphospholipid syndromes," the interaction of exposed plasma membrane PS and PE with plasma proteins is now generally believed to induce offending antigens (M. D. Smirnov, et al., J. Clin. Invest. 95: 309-316, 1995).
Relationship of PS Exposure to Programmed Cell Death
Programmed cell death (apoptosis) is now recognized to be central to the selective elimination of mammalian cells during embryogenesis, tissue re-modeling, and in the clonal selection of immune cells (P. D. Allen, et al., Blood Rev. 7:63-73, 1993; J. J. Cohen, Immunol. Today 14:126-130, 1993). The apoptotic cell undergoes characteristic changes, including elevated [Ca.sup.2+ ].sub.i, altered phospholipid packing, surface exposure of PS, plasma membrane blebbing and vesiculation, cell shrinkage, chromatin condensation, nucleolar desintegration, and at late stages, DNA degradation by Ca.sup.2+ /Mg.sup.2+ -dependent endonuclease(s), with characteristic fragmentation into 180 bp multimers ("DNA laddering"). The transcriptional events that initiate apoptosis remain unresolved, but evidence implicates certain proto-oncogenes, including c-myc as activators, and other proto-oncogenes, including bcl-2, as suppressors (P. D. Allen, et al., supra, 1993; J. C. Reed, J. Cell. Biol. 124:1-6, 1994). In thymocytes and B-lymphocytes, an apoptotic transformation can be induced by dexamethasone (activating glucocorticoid receptors) and by cAMP (protein kinase A pathway) (D. J. McConkey, et al., J. Immunol. 145:1227-1230, 1990; N. Kaiser, et al., Proc. Natl. Acad. Sci. USA 74:638-642, 1977; J. J. Cohen, et al., J. Immunol. 132:38-42, 1984; R. Merino, et al., EMBO J. 13:683-691, 1994; M. K. Newell, et al., Proc. Natl. Acad. Sci. USA 90:10459-10463, 1993), as well as directly through treatment with Ca.sup.2+ ionophore (Z.-Q. Ning, et al., Eur. J. Immunol. 23:3369-3372, 1993), implicating [Ca.sup.2+ ].sub.i as a central mediator of the cellular changes that accompany apoptosis. The similarity of the plasma membrane changes noted for apoptotic cells, to those elicited by elevation of [Ca.sup.2+ ].sub.i in platelets, erythrocytes, and other cells that do not undergo apoptosis, suggest that the nuclear and plasma membrane changes associated with apoptosis are separate "epiphenomena", reflecting independent and unrelated responses to a coordinate rise in [Ca.sup.2+ ].sub.i.
Diaz, et al. (Blood 87[7]:2956-2961, 1996) have recently reported the generation of phenotypically aged phosphatidylserine-expressing erythrocytes by dilauroylphosphatidylcholine (DLPC)-induced vesiculation. Red blood cells were artificially vesiculated with DLPC and assessed for alterations in density, membrane lipid asymmetry and propensity to be recognized by macrophages in vitro and the reticuloendothelial system in vivo. The results suggest that vesiculation contributes to alterations is membrane lipid asymmetry and cell characteristics of the aged red blood cell phenotype.
Role of Cell Surface PS in Clearance by the RE System
There is now accumulating data to suggest that cell-surface PS may contribute to the recognition and clearance of senescent, injured, or apoptotic cells by macrophages and other cells of the reticuloendothelial system (J. Savill, et al., Immunol. Today 14:131-136, 1993; V. A. Fadok, et al., J. Immunol. 148:2207-2216, 1992; J. Connor, et al., J. Biol. Chem. 269:2399-2404, 1994). These experiments demonstrate that (i) macrophages have inducible receptors that stereospecifically bind to PS-containing liposomes and to surface-exposed plasma membrane PS; (ii) selective phagocytosis of apoptotic lymphocytes by stimulated macrophages is observed in the absence of plasma proteins, and this can be inhibited by PS-containing liposomes or by phosphoserine (V. A. Fadok, et al., supra, 1992). Consistent with these data, the circulating lifetime of infused PS-containing liposomes is markedly decreased when compared to liposomes devoid of PS, due to rapid hepato-splenic clearance (T. M. Allen, et al., Proc. Natl. Acad. Sci. USA 85:8067-8071, 1988). Similarly, increased exposure of plasma membrane PS during in vitro storage of platelet concentrates may contribute to an accelerated clearance of these cells after transfusion (A. P. Bode, et al., Thromb. Res. 39:49-61, 1985; A. P. Bode, et al., J. Lab. Clin. Med. 113:94-102, 1989; A. P. Bode, et al., Blood 77:887-895, 1991; D. Geldwerth, et al., J. Clin. Invest. 92:308-314, 1993; P. Gaffet, et al., Eur. J. Biochem. 222:1033-1040, 1994; E. M. Bruckheimer, et al., J. Leukoc. Biol. 59, 784-788, 1996; C. Diaz, et al, supra, 1996). This possibility is underscored by recent reports documenting increased PS exposure in platelets and red cells during in vitro storage. Evidence that PS exposed on the surface of tumor cells promotes adherence and cytolysis by inflammatory macrophages has also been reported (J. Connor, et al., Proc. Natl. Acad. Sci. USA 86:3184-3188, 1989).
Regulation of the Transmembrane Distribution of PS
It is now well established that phospholipids are normally asymmetrically distributed within the plasma membrane of all blood cells, vascular endothelium, and other cells: the aminophospholipids (including phosphatidylserine (PS) and phosphatidylethanolamine (PE)) reside almost exclusively in the inner membrane leaflet, whereas the outer leaflet is enriched in neutral polar phospholipids, including phosphatidylcholine (PC) and sphingomyelin (B. Roelofsen, Infection 19:S206-S209, 1992; A. J. Schroit, et al., Biochim. Biophys. Acta 1071:313-329, 1991; P. F. Devaux, Biochemistry 30:1163-1173, 1991). It is well-recognized that the transmembrane orientation of plasma membrane PL is central to the regulation of surface-localized enzyme reactions of both complement and coagulation systems and to the recognition and phagocytic clearance of injured, aged or apoptotic cells. It is also now generally accepted that the maintenance of PL asymmetry arises through the activity of a specific transmembrane PL "flippase" with specificity for aminoPL. This aminoPL translocase (APT) has been shown to selectively and vectorially transport PS (&gt;PE), but not neutral PL such as PC, from outer to inner leaflets of the plasma membrane in a process that is dependent on both Mg.sup.2+ and ATP, inhibited by fluoride, o-vanadate or increased [Ca.sup.2+ ].sub.c, and inactivated by N-ethylmaleimide (NEM) or pyridyldithioethylamine (PDA)( M. Bitbol, et al., Biochim. Biophys. Acta 904:268-282, 1987; M. Seigneuret, et al., Proc. Natl. Acad. Sci. USA 81:3751-3755, 1984; J. Connor, et al., Biochemistry 26:5099-5105, 1987; P. F. Devaux, et al., Phys. Lipids 73:107-120, 1994; A. Zachowski, et al., Biochemistry 25:2585-2590, 1986; C. Diaz, et al., supra, 1996). In addition to plasma membrane, APT activity has also been identified in the membranes of secretory vesicles and synaptosomes (A. Zachowski, et al., Nature 340:75-76, 1989). The K.sub.m for ATP is approximately 1 mM, and it has been estimated that one molecule of ATP is hydrolyzed for each aminoPL transported (Z. Beleznay, et al., Biochemistry 32:3146-3152, 1993). Two candidate proteins have been proposed to function as APT: the Rh antigen protein, and a 110-120 kDa Mg.sup.2+ -ATPase. Schroit and coworkers (A. J. Schroit, et al., Biochemistry 29:10303-10306, 1990) originally proposed that a 32 kDa PS-binding RBC membrane protein that precipitated with antibody to Rh was the erythrocyte APT. Subsequently it was shown that Rh.sub.null cells deficient in Rh antigen nevertheless exhibit normal APT activity, and very recently, the 32 kDa PS-binding protein that co-precipitates with Rh protein was identified as stomatin, and it was shown that its interaction with PS was not specific for the aminoPL headgroup. This implies that neither stomatin, nor, the Rh protein can provide APT function (J. Desneves, et al., Biophys. Res. Commun. 224:108-114, 1996). The observed similarity in cation, ATP- and PS-dependence of cellular APT activity to a partially purified Mg.sup.2+ -dependent ATPase from RBC led Devaux and associates, and later Daleke, to suggest that APT is a specific Mg.sup.2+ -ATPase (A. Zachowski, et al. supra, 1989; G. Morrot, et al., FEBS Lett. 266:29-32; D. L. Daleke, et al., Ann. NY Acad. Sci. 671:468-470, 1992; M. L. Zimmerman, et al., Biochemistry 32:12257-12263, 1993). Consistent with this premise, Auland (Auland, et al., Proc. Natl. Acad. Sci 91:10938-10942, 1994) demonstrated PS-specific transport in proteoliposomes reconstituted with an unidentified Mg.sup.2+ -ATPase isolated from RBC. Recently, an ATPase II from bovine chromaffine granules has been cloned and sequenced, and evidence has been presented that this enzyme may exhibit aminoPL translocase activity (X. J. Tang, et al., Science 272:1495-1497, 1996).
Ca.sup.2+ and the Collapse of Phospholipid Asymmetry
Whereas the rate of spontaneous flip/flop of PL between membrane leaflets is normally quite slow, a substantial rise in [Ca.sup.2+ ].sub.c resulting from agonist-induced activation, programmed cell death, or, secondary to immune injury, initiates rapid transbilayer migration of all plasma membrane PL with net movement of aminoPL to the outer leaflet, collapsing the normal asymmetric distribution (P. Williamson, et al., Biochemistry 31:6355-6360, 1992); F. Basse, et al., Biochemistry 32:2337-2344, 1993; C.-P. Chang, et al., supra, 1993; P. Comfurius, et al. Biochim. Biophys. Acta 1026:153-160, 1990; A. J. Schroit, supra, 1991; P. Devaux, Biochemistry 30:1163-1173, 1991; J. Connor, et al., J. Biol. Chem. 267:19412-19417, 1992). Four different mechanisms have been proposed to account for this induced "scrambling" of plasma membrane PL with net egress of aminoPL to cell surfaces (A. J. Schroit, supra, 1991; P. Devaux, supra, 1992; P. Devaux, supra, 1991; R. F. A. Zwaal, et al., Biochim. Biophys. Acta 1180:1-8, 1992): (i) spontaneous collapse of PL asymmetry due to inactivation of vectorial transport by plasma membrane APT; (ii) random scrambling due to transient formation of non-bilayer (H.sub.II -phase) PL domains upon Ca.sup.2+ -induced blebbing of plasma membrane vesicles; (iii) direct effects of Ca.sup.2+ on topology and distribution of anionic PLs; (iv) response of a Ca.sup.2+ -sensitive protein(s) that facilitates PL transfer between membrane leaflets.
(i) Spontaneous Collapse of PL Asymmetry
APT is Inhibited at elevated [Ca.sup.2+ ].sub.c, raising the possibility that the concomitant egress of aminoPL to the cell surface simply reflects spontaneous back-leak ("flop") of the PL distribution that is constitutively maintained by APT (P. Williamson, et al., supra, 1992; P. F. Devaux, supra, 1992; P. Devaux, supra, 1991). Nevertheless, inhibition of APT--either by depletion of cellular ATP, or by incubation with fluoride, o-vanadate or NEM--does not in itself cause accelerated transbilayer PL migration or significant cell-surface PS exposure, as long as normally low [Ca.sup.2+ ].sub.c is maintained (E. M. Bevers, et al., supra, 1991; P. Comfurius, et al. supra, 1990; B. Verhoven, et al., Biochim. Biophys. Acta 1104:15-23, 1992; J. Connor, et al. Biochemistry 29:37-43, 1990). Conversely, cells genetically deficient in PL scramblase show normal APT activity. Thus it appears that the spontaneous transbilayer migration of plasma membrane PL is inherently very slow at the normal low [Ca.sup.2+ ].sub.c, whereas entry of Ca.sup.2+ into the cytosol specifically induces rapid movement of PL between plasma membrane leaflets. Whereas the inherent rate of transbilayer migration of PL cannot account for the rapid scrambling observed at elevated [Ca.sup.2+ ].sub.c, it is conceivable that interaction of [Ca.sup.2+ ].sub.c with APT induces a conformational change that not only inactivates the ATP-dependent inward translocation of aminoPL, but also facilitates selective flop of PS & PE to the outer leaflet. In this context, Bienvenue and associates (Basse, et al, supra, 1993; Gaffet, et al., Biochemistry 34:6762-6769, 1995) have reported evidence for transient vectorial egress of PS upon elevation of [Ca.sup.2+ ].sub.c in platelet, whereas data from others suggest bidirectional and non-selective transbilayer scrambling of all plasma membrane PL (including PC which is not flipped by APT) at elevated [Ca.sup.2+ ].sub.c (P. Williamson, et al., supra, 1992; P. Williamson, et al., Biochemistry 34:10448-10455, 1995; E. F. Smeets, et al., Biochim. Biophys. Acta Bio-Membr. 1195:281-286, 1994; D. Bratton, J. Biol. Chem. 269:22517-22523, 1994).
(ii) Relationship of PS Egress to Shedding of Plasma Membrane Vesicles
Data from our laboratory helped establish that surface exposure of PS is intimately related to a process of Ca.sup.2+ -induced vesiculation of the plasma membrane, and that formation of such PS-rich plasma membrane "microparticles" contributes to expression of cellular procoagulant activity (C.-P. Chang, et al., supra, 1993; P. J. Sims, et al., supra, 1989; A. P. Bode, et al., supra, 1985; R. F. A. Zwaal, et al., supra, 1992; T. Wiedmer, et al., supra, 1990; P. J. Sims, et al., J. Biol. Chem. 263:18205-18212, 1988; K. K. Hamilton, et al., J. Biol. Chem. 265:3809-3814, 1990; H. Sandberg, et al., Thromb. Res. 39:63-79, 1985). This correlation between microparticle formation and surface exposure of PS suggested that the membrane fusion events generating these membrane vesicles underlie observed scrambling of plasma membrane PL, presumably through transient formation of H.sub.II -phase PL (C. P. Chang, et al., supra, 1993). Alternatively, prior egress of PS to the outer leaflet might create a mass imbalance that itself drives plasma membrane evagination and vesiculation (P.F. Devaux, supra, 1991). In this context, we and others have observed that PS migration to the cell surface can precede membrane vesiculation, and can occur without microparticle formation (G. E. Gilbert, et al., J. Biol. Chem 266:17261-18269, 1991; F. Basse, et al., Biochemistry 32:2337-2344, 1993; P. Gaffet, et al., supra, 1995)). Our data suggested the participation of a calmodulin-dependent kinase in Ca.sup.2+ -induced vesiculation (T. Wiedmer, et al., Blood 78:2880-2886, 1991). Involvement of a protein kinase in the cytoskeletal reorganization required for platelet microparticle formation has recently been confirmed (Y. Yano, et al. Biochem. J. 298:303-308, 1994). This suggests that whereas membrane fusion may contribute, it neither initiates nor is required for PL scramblase function.
(iii) Interaction of Ca.sup.2+ With Anionic Plasma Membrane PL
Ion pairing of Ca.sup.2+ with the anionic PS headgroup might accelerate transbilayer migration by lowering the energy barrier to crossing through the hydrophobic membrane interior. However, Ca.sup.2+ does not directly induce transbilayer migration of PL in PS-containing membranes, except when mole % of PS is sufficient to induce an H.sub.II -phase and vesicle-vesicle fusion (A. L. Bailey, et al., Biochemistry 33:12573-12580, 1994; B. de Kruijff, et al., Trends Biochem. Sci. 5:79-81, 1980). Thus ion pairing of Ca.sup.2+ with inner leaflet PS would not appear to directly induce PL flip/flop between membrane leaflets. Alternatively, Devaux and associates (J.-C. Sulpice, et al., J. Biol. Chem. 269:6347-6354, 1994; J.-C. Sulpice, et al., Biochemistry 35:13345-13352, 1996) proposed that Ca.sup.2+ binding to phosphatidylinositol 4,5-bisphosphate (PIP2) induces transbilayer migration of other PL, based on the observation that adding PIP2 to RBC promoted Ca.sup.2+ -dependent transbilayer movement of PS. Nevertheless, subsequent studies revealed that this incorporation of exogenous PIP2 also induces enough membrane lysis to account for any apparent increase in transbilayer migration of PS (Bevers, et al. Blood 86:1983-1991, 1995).
(iv) Role of Ca.sup.2+ -Sensitive Protein(s) in PL Redistribution
Elevation of [Ca.sup.2+ ].sub.c is known to give rise to marked changes in several cytoskeletal and membrane proteins that might affect the rate of movement of PL between plasma membrane leaflets. For example, several cytoskeletal proteins (including, spectrin and erythrocyte band 4.1) (A. M. Cohen, et al., Blood 68:920-926, 1986; S. B. Sato, et al., Eur. J. Biochem. 130:19-25, 1983; A. C. Rybicki, et al., J. Clin. Invest. 81:255-260, 1988; K. A. Shiffer, et al., Biochim. Biophys. Acta 937:269-280, 1988) have been shown to bind specifically to the PS headgroup, and this interaction potentially serves to trap PS in the inner leaflet of the plasma membrane (P. Comfurius, et al., Biochim. Biophys. Acta 983:212-216, 1989). Breaking such interactions, as might occur through interaction of Ca.sup.2+ with the PS headgroup, or through proteolytic degradation of cytoskeletal proteins, would potentially dissociate PS from these endofacial contacts and thereby accelerate PS flop to the outer leaflet (P. F. Franck, et al., supra, 1985). In this context, it has been reported that polyamines inhibit the endogenous PL scramblase activity of the RBC membrane, suggesting that these polycations inhibit interaction of Ca.sup.2+ at its endofacial membrane site (D. L. Bratton, et al., supra, 1994; J.-C. Sulpice, et al. supra, 1996). Calpain-mediated proteolysis of components of the submembrane cytoskeleton can be temporally-correlated to membrane vesiculation and to surface exposure of PS. Nevertheless, inhibition of calpain does not prevent PS egress, and, Ca.sup.2+ -dependent PL scramblase activity is readily demonstrated in resealed RBC ghosts and inside-out RBC membrane vesicles (IOV) washed free of calpains and other soluble proteases (P. Comfurius, et al., supra, 1990; T. Wiedmer, et al., Biochemistry 29:623-632, 1990; Fox, et al., J. Biol. Chem. 266:13289-13295, 1991; J. E. B. Fox, et al., J. Cell Biol. 111:483-493, 1990; P. F. J. Verhallen, et al., Biochim. Biophys. Acta 903:206-217, 1987; L. Dachary-Prigent, et al., Blood 81:2554-2565, 1993) On the other hand, the possibility that a membrane protein with inherent PL scramblase activity directly mediates Ca.sup.+ -induced transbilayer migration of PL was suggested by the observation that this activity is inhibited by sulfhydryl oxidation of membrane proteins with PDA (P. Williamson, et al., supra, 1995). Consistent with this finding, we recently reported the purification and preliminary characterization of an integral RBC membrane protein that, when reconstituted in liposomes, mediates a Ca.sup.2+ -dependent transbilayer movement of PL mimicking plasma membrane PL reorganization evoked upon elevation of [Ca.sup.2+ ].sub.c (F. Basse, et al., J. Biol. Chem. 271:17205-17210, 1996) Evidence that a protein of similar function must also be present in platelets was recently reported by Zwaal (P. Comfurius, et al., Biochemistry 35:7631-7634, 1996).
The Scott Syndrome
Scott syndrome is a bleeding disorder described in a single patient that reflects impaired expression by activated platelets of the membrane sites that are required for normal assembly of the prothrombinase and tenase enzyme complexes (H. J. Weiss, et al., Am. J. Med. 67:206-213, 1979; H. J. Weiss, Semin. Hematol. 31:1-8, 1994). Platelets obtained from this patient secrete and aggregate normally when exposed to various agonists (ADP, thrombin, collagen, complement proteins C5b-9, or calcium ionophore), but when fully activated, exhibit a marked deficiency of membrane binding sites for factors Va and VIIIa, reflecting a concomitant reduction in the amount of surface-exposed PS (J. P. Miletich, et al., supra, 1979; J. Rosing, et al., supra, 1985; J. P. Sims, et al., supra, 1989; S. S. Ahmad, et al., supra, 1989).
Although Scott syndrome was originally described as an isolated platelet disorder, it is now clear that other blood cells from this patient, including erythrocytes and lymphocytes, are affected as well (E. M. Bevers, et al. Blood 79:380-388, 1992). Measurements that have been made by ourselves and others suggest that Scott platelets and erythrocytes contain normal amounts of PS and other phospholipids, and also exhibit normal aminophospholipid translocase activity (H. J. Weiss, et al., 1979). These cells are defective, however, in their capacity to mobilize PS from inner to outer membrane leaflets i n response to elevated [Ca.sup.2+ ].sub.i, a response that is now thought to be required for normal prothrombinase and tenase assembly. A search for the molecular defect responsible for the Scott syndrome has to date failed to reveal abnormality in platelet or red cell proteins (probed by 2-dimensional electrophoresis); the agonist-induced elevation of [Ca.sup.+ ] in Scott platelets is normal; and the calpain and transglutaminase activities of both Scott platelets and RBCs are indistinguishable from normal controls, as assessed by the Ca.sup.2+ -induced cleavage or cross-linking of cytoskeletal proteins (P. Comfurius, et al., Biochim. Biophys. Acta 815:143-148, 1985).
We have established in in vitro culture EBV-transformed lymphoblast cell lines from this patient and have demonstrated that these immortalized cells exhibit the same phenotype of impaired Ca.sup.2+ -induced plasma membrane phospholipid scrambling that is characteristic of the platelets and erythrocytes in this disorder. Our data also establish that this defect propagates through multiple cell divisions and can be corrected by heterokaryon fusion with wild-type cells that exhibit normal plasma membrane phospholipid scrambling (H. Kojima, et al. J. Clin. Invest. 94:2237-2244, 1994).
Similar data have recently been reported for a second patient with Scott syndrome, and evidence for a familial inheritance of the cellular defect provided (F. Toti, et al., supra, 1996)). This implies that the molecular basis for this clinical disorder relates to a gene defect that results in impaired activity of a cellular protein that is expressed in a variety of cell lineages, and that this protein normally mediates the intracellular Ca.sup.2+ -dependent transbilayer movement (or "scrambling") of plasma membrane phospholipids that occurs in response to cell activation, apoptosis, or cell injury. We identify this protein as "phospholipid scramblase", "PL scramblase", and "P37". We mean for "P37" to be synonymous with "phospholipid scramblase or PL scramblase" and refer to these names interchangeably throughout the text.
The loss of normal PL scramblase function in patients with Scott syndrome may relate to either the abnormal expression of an inhibitor of the activity of PL scramblase, a deletion or loss of function mutation in the gene encoding P37 protein, or, a mutation affecting a cofactor of P37 that is required for normal expression of its PL scramblase activity.
Patients with the Scott syndrome defect display abnormal bleeding and a prolongation of the time required for blood clotting (H. J. Weiss, Semin. Hematol. 31:1-8, 1994). This implies that activation of PL scramblase is normally required for effective clot formation and for efficient hemostasis, whereas loss or inhibition of PL scramblase activity leads to retarded blood clotting. We therefore propose that the selective activation of PL scramblase function is of potential therapeutic value in the acceleration of hemostasis and in preventing blood loss, whereas the selective inhibition of PL scramblase function is of potential therapeutic value in certain thrombotic disorders characterized by excessive or inappropriate clot formation due to expression of plasma membrane procoagulant activity.
In this application, we identify the cellular component that functions to mediate the Ca.sup.2+ -dependent reorganization of plasma membrane phospholipids and describe methods for preventing egress of PS to the surface of activated, injured, or apoptotic cells.